The primary objective of this project is to define molecular and biochemical mechanisms involved in eukaryotic DNA synthesis. To approach this problem, we are investigating adeno-associated virus (AAV) DNA replication in both in vivo and in vitro systems. We have shown that AAV DNA synthesis can be initiated in vitro in cell-free extracts and that replicating forms that correspond to those identified in vivo can be synthesized with either endogenous or exogenously added templates. Recently, we have shown that AAV "rep" proteins may not be required for the synthesis of AAV concatemeric DNA intermediates, but that one on more of these proteins is required to resolve the long intermediates into unit length duplexes. The observed mode of AAV DNA replication may serve as a model for telomere replication in eukaryotes. Among methods used are affinity chromatography, DNA sequence analysis, and restriction cleavage of DNA molecules and DNA transfection.